Plants synthesizing a modified starch, a process for the generation of the plants, their use, and the modified starch

ABSTRACT

The present invention relates to recombinant nucleic acid molecules which contain two or more nucleotide sequences which encode enzymes which participate in the starch metabolism, methods for generating transgenic plant cells and plants which synthesize starch which is modified with regard to its phosphate content and its side-chain structure. The present invention furthermore relates to vectors and host cells which contain the nucleic acid molecules according to the invention, the plant cells and plants which originate from the methods according to the invention, to the starch synthesized by the plant cells and plants according to the invention, and to processes for the preparation of this starch.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/424,799, filed Apr. 28, 2003 now U.S. Pat. No. 7,247,769, which is acontinuation of U.S. patent application Ser. No. 09/364,185, filed Jul.29, 1999, now U.S. Pat. No. 6,596,928, which claims priority to Germanpatent application DE 19836098.3, filed Jul. 31, 1998, the disclosuresof each of which are hereby incorporated by reference.

FIELD OF INVENTION

The present invention relates to recombinant nucleic acid moleculeswhich comprise two or more nucleotide sequences which encode enzymeswhich participate in the starch metabolism, to processes for generatingtransgenic plant cells, and plants which synthesize starch which ismodified with regard to their phosphate content and their side-chainstructure. The present invention furthermore relates to vectors and hostcells which contain the nucleic acid molecules according to theinvention, the plant cells and plants which originate from the processesaccording to the invention, to the starch synthesized by the plant cellsand plants according to the invention, and to processes for thepreparation of this starch.

BACKGROUND OF THE INVENTION

Bearing in mind the increasing importance of plant constituents asrenewable resources, biotechnology research attempts to adapt plant rawmaterials to the demands of the processing industry. Thus, to makepossible the use of renewable resources in as many fields of applicationas possible, it is necessary to make available a great variety ofmaterials.

Not only oils, fats and proteins, but also polysaccharides, constituteimportant renewable resources from plants. A pivotal position in thepolysaccharides is taken up not only by cellulose, but also by starch,which is one of the most important storage substances in higher plants.Not only corn, rice and wheat, but also potato, plays an important role,in particular in starch production.

The polysaccharide starch is a polymer of chemically uniform units, theglucose molecules. However, it is of a highly complex mixture ofdifferent forms of molecules which differ with regard to their degree ofpolymerization and the occurrence of branchings in the glucose chains.Starch is therefore no uniform raw material. In particular, wedifferentiate between amylose starch, an essentially unbranched polymerof α-1,4-glycosidically linked glucose molecules, and amylopectin starchwhich, in turn, constitutes a complex mixture of differently branchedglucose chains. Other branchings are generated by the occurrence ofadditional α-1,6-glycosidic linkages. In typical plants used for starchproduction such as, for example, corn or potatoes, the synthesizedstarch consists to approx. 25% of amylose starch and approx. 75% ofamylopectin starch.

The molecular structure of the starch, which is largely determined bythe degree of branching, the amylose/amylopectin ratio, the averagelength and distribution of the side chains and the presence of phosphategroups is of prime importance for important functional properties of thestarch, resp., its aqueous solutions. Important functional propertieswhich must be mentioned are, for example, solubility, retrogradationbehavior, film-forming properties, viscosity, color stability, thegelatination properties and the binding and adhesion properties. Also,starch granule size may be of importance for various applications. Theproduction of high-amylose starches is also of particular interest forcertain applications. Furthermore, a modified starch contained in plantcells may advantageously alter the behavior of the plant cells undercertain conditions. For example, a reduced starch degradation during thestorage of starch-containing organs such as, for example, seeds ortubers, before their processing, for example for starch extraction, isfeasible. It is also of interest to produce modified starches which makeplant cells or plant organs which contain this starch better suited forprocessing, for example in the preparation of foodstuffs such as popcornor cornflakes from corn or of potato chips, potato crisps or potatopowder from potatoes. Of particular interest is the improvement of thestarches regarding a reduced cold sweetening, i.e. a reduced liberationof reducing sugars (in particular glucose) upon prolonged storage at lowtemperatures. Potatoes especially are frequently stored at temperaturesfrom 4 to 8° C. to minimize starch degradation during storage. Thereducing sugars liberated during this process, in particular glucose,result in undesired browning reactions in the production of potato chipsor potato crisps (so-called Maillard reactions).

The starch which can be isolated from plants is frequently adapted toparticular industrial purposes with the aid of chemical modificationswhich, as a rule, require time and money. It seems therefore desirableto find possibilities of generating plants which synthesize a starchwhose properties already meet the specific demands of the processingindustry and thus combine economical and ecological advantages.

A possibility of providing such plants is, in addition to breedingmeasures, the direct genetic alteration of the starch metabolism ofstarch-producing plants by genetic engineering methods. However, aprerequisite therefor is the identification and characterization of theenzymes which participate in starch synthesis modification and starchdegradation (starch metabolism) and isolation of the corresponding DNAsequences which encode these enzymes.

The biochemical pathways which lead to the synthesis of starch areessentially known. In plant cells, starch synthesis takes place in theplastids. In photosynthetically active tissues, these plastids are thechloroplasts, in photosynthetically inactive, starch-storing tissues theamyloplasts.

Important enzymes which participate in starch synthesis are, forexample, the branching enzymes, ADP glucose pyrophosphorylases,granule-bound starch synthases, soluble starch synthases, debranchingenzymes, disproportioning enzymes, plastidic starch phosphorylases andthe R1 enzymes (R1 proteins).

OBJECTS AND SUMMARY OF THE INVENTION

It is an object of the present invention to provide other, oralternative, genetic engineering methods for modifying the starchmetabolism in starch-synthesizing plants (for example rye, barley, oats,corn, wheat, sorghum and millet, sago, rice, peas, marrowfat peas,cassava, potatoes, tomatoes, oilseed rape/canola, soybeans, hemp, flax,sunflowers, cowpeas, mung beans, beans, bananas or arrowroot) orsuitable nucleic acid molecules by means of which plant cells can betransformed to allow the synthesis of altered advantageous starchvarieties.

Such altered starch varieties exhibit, for example, modificationsregarding their degree of branching, the amylose/amylopectin ratio, thephosphate content, the starch granule size and/or the average length anddistribution of the side chains (i.e. the side chain structure).

It is a further object of the invention to provide methods which allowthe generation of transgenic plants which synthesize an altered(modified) starch variety.

Surprisingly, transgenic plant cells or plants which have beentransformed with the nucleic acid molecules according to the inventionsynthesize a starch which is altered in the particular manner accordingto the invention with regard to its physicochemical properties and/orits side chain structure. In contrast, known starches which weresynthesized by transgenic plants do not exhibit alterations according tothe invention.

These objects are achieved according to the invention by providing theuse forms specified in the claims.

DETAILED DESCRIPTION OF THE INVENTION

The invention therefore relates to a recombinant nucleic acid molecule(nucleotide sequence) comprising a) at least one nucleotide sequence(polynucleotide, resp., nucleic acid molecule) encoding a protein havingthe function of a soluble starch synthase III or fragments of saidnucleotide sequence and b) one or more nucleotide sequences which encodea protein selected from the group A, consisting of proteins having thefunction of branching enzymes, ADP glucose pyrophosphorylases,granule-bound starch synthases, soluble starch synthases I, II, orother, debranching enzymes, disproportioning enzymes, plastidic starchphosphorylases, R1 enzymes, amylases, and glucosidases, or fragmentsthereof—preferably, soluble starch synthases I, soluble starch synthasesII and/or branching enzymes or fragments thereof—and nucleic acidmolecules which hybridize with one of the said nucleotide sequences orfragments thereof, preferably a deoxyribonucleic acid molecule orribonucleic acid molecule, especially preferably a cDNA molecule.Especially preferred is a nucleic acid molecule which hybridizesspecifically with one of said nucleotide sequences or fragments thereof.

Nucleotide sequences which are suitably employed according to theinvention and which encode a protein having the function of solublestarch synthase III are disclosed, for example, in EP-A-0779363 and Abelet al., 1996, Plant J. 10(6):981-991 (SEQ ID NO:1). The term “nucleotidesequence encoding a protein having the function of a soluble starchsynthase III” is to be understood as meaning for the purposes of thepresent invention in particular those sequences whose encoding regionhas a length of 3000-4500 bp, preferably 3200-4250 bp, especiallypreferably 3400-4000 bp and whose homology to the entire encoding regionof a nucleic acid encoding a protein which has the function of a starchsynthase (SEQ ID NO: 1) amounts to at least 70%, preferably at least80%, especially preferably at least 90% and very especially preferablyat least 95%.

Nucleotide sequences which are suitable according to the invention andwhich encode a protein from the group A are, for example, soluble starchsynthases (Type I, II or other) or granule-bound starch synthaseisoforms (e.g., Hergersberg, 1988, PhD thesis, University of Cologne;Abel, 1995, PhD thesis. FU Berlin; Abel et al., 1996, Plant Journal10(6):981-991; Visser et al., 1989, Plant Sci. 64:185-192; van der Leijet al., 1991, Mol. Gen. Genet. 228:240-248; EP-A-0779363; WO 92/11376;WO 96/15248, wherein SSSB has the meaning of soluble starch synthase I,and GBSSII has the meaning of soluble starch synthase II; WO 97/26362;WO 97/44472; WO 97/45545; Delrue et al., 1992. J. Bacteriol. 174:3612-3620; Baba et al., 1993, Plant Physiol. 103:565-573; Dry et al.,1992, The Plant Journal 2,2: 193-202 or else in the EMBL databaseentries X74160; X58453; X88789); branching enzyme isoforms (branchingenzymes I, IIa, IIb), debranching enzyme isoforms (debranching enzymes,isoamylases, pullulanases) or disproportioning enzyme isoforms,described, for example, in WO 92/14827; WO 95/07335; WO 95/09922; WO96/19581; WO 97/22703; WO 97/32985; WO 97142328; Takaha et al., 1993, J.Biol. Chem. 268: 1391-1396 or else in the EMBL database entry X83969,and those for ADP glucose pyrophosphorylases, plastidic starchphosphorylase isoforms and R1 enzymes (R1 proteins), described, forexample, in EP-A-0368506; EP-A-0455316; WO 94/28146; DE 1963176.4; WO97/11188; Brisson et al., 1989, The Plant Cell 1:559-566; Buchner etal., 1996, Planta 199:64-73; Camirand et al., 1989, Plant Physiol. 89(4Suppl.) 61; Bhatt & Knowler, J. Exp. Botany 41 (Suppl.) 5-7; Lin et al.,1991, Plant Physiol. 95:1250-1253; Sonnewald et al., 1995, Plant Mol.Biol. 27:567-576; DDBJ No. D23280; Lorberth et al., 1998, NatureBiotechnology 16:473-477, and amylases and glucosidases

The nucleotide sequences which are suitably employed in accordance withthe invention are of prokaryotic or eukaryotic origin, preferably ofbacterial, fungal or plant origin.

The term “fragment” denotes, for the purposes of the present invention,portions of the nucleic acid molecule according to the invention or of anucleic acid molecule to be suitably employed in accordance with theinvention which has a length of at least 15 bp, preferably at least 150bp, especially preferably at least 500 bp, but which generally do notexceed a length of 5000 bp, preferably 2500 bp. In particular, the term“fragments” encompasses biologically active molecules.

The term “hybridization” denotes, for the purposes of the presentinvention, a hybridization under conventional hybridization conditions,preferably under stringent conditions as they are described, forexample, by Sambrook et al., Molecular Cloning, A Laboratory Manual, 2ndEdition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.).

Especially preferably, a “specific hybridization” takes place under thefollowing highly stringent conditions:

Hybridization buffer: 2×SSC; 10×Denhardt solution (Ficoll 400+PEG+BSA;ratio 1:1:1); 0.1% SDS; 5 mM EDTA; 50 mM Na₂HP0₄; 250 μg/ml herringsperm DNA; 50 μg/ml tRNA; or 0.25 M sodium phosphate buffer pH 7.2; 1 mMEDTA; 7% SDS at a

-   Hybridization temperature of T=55 to 68° C.,-   Wash buffer: 0.2×SSC; 0.1% SDS and-   Wash temperature: T=40 to 68° C.

The molecules which hybridize with the nucleic acid molecules accordingto the invention or with the nucleic acid molecules to be suitablyemployed in accordance with the invention also encompass fragments,derivatives and allelic variants of the nucleic acid molecules accordingto the invention or to be suitably employed in accordance with theinvention. “Fragments” are not only to be understood as portions of thenucleic acid molecules which are long enough to encode a functionallyactive portion of the proteins described. The term “derivative” means,within the context of the present invention, that the sequences of thesemolecules differ from the sequences of the nucleic acid moleculesaccording to the invention or to be suitably employed in accordance withthe invention in one or more positions and exhibit a high degree ofhomology to these sequences. Homology means a sequential identity of atleast 60%, preferably over 70%, and especially preferably over 85%, inparticular over 90% and very especially preferably over 95%. Thedeviations relative to the nucleic acid molecules according to theinvention or to the nucleic acid molecules to be suitably employed inaccordance with the invention may have originated by means of one ormore deletions, substitutions, insertions (addition) or recombinations.

Furthermore, homology means that a functional and/or structuralequivalence exists between the nucleic acid molecules in question andthe proteins encoded by them. The nucleic acid molecules which arehomologous to the molecules according to the invention or to themolecules to be suitably employed in accordance with the invention andwhich constitute derivatives of these molecules are, as a rule,variations of these molecules which constitute modifications which exertthe same, a virtually identical or a similar biological function. Theymay be naturally occurring variations, for example sequences from otherplant species or mutations, it being possible for these mutations tohave occurred naturally or to have been introduced by naturalmutagenesis. The variations may further be synthetic sequences. Theallelic variants may be naturally occurring variants or else syntheticvariants or variants generated by recombinant DNA technology.

The nucleic acid molecules according to the inventor or to be suitablyemployed in accordance with the invention may be DNA molecules, inparticular cDNA molecules, or, if appropriate, the combination ofgenomic molecules. The nucleic acid molecules according to the inventionor to be suitably employed in accordance with the invention mayfurthermore be RNA molecules. The nucleic acid molecules according tothe invention or to be suitably employed in accordance with theinvention or fragments thereof may have been obtained, for example, fromnatural sources, generated by means of recombinant technology orgenerated by synthesis.

To express the nucleic acid molecules according to the invention or thenucleic acid molecules to be suitably employed in accordance with theinvention in sense or antisense orientation in plant cells. Theseinclude in general, promoters. In general, any promoter which is activein plant cell is suitable for expression. The promoter may have beenchosen to in such a way that expression is constitutive or only in aparticular tissue, at a particular point in time of plant development orat a point in time determined by external factors which can be, forexample, chemically or biologically inducible. Relative to thetransformed plant, the promoter can be homologous or heterologous, ascan be the nucleotide sequence. Examples of suitable promoters are thecauliflower mosaic virus 35S RNA promoter and the ubiqutin-promotorderived from corn for constitutive expression, the palatin promoter B33(Rocha-Sosa et al., 1989, EMBO J., 8:23-29) for tuber-specificexpression in potatoes or a promoter which ensures expression only inphotosynthetically active tissues, for example the ST-LS1 promoter(Stockhaus et al., 1987,Proc. Natl. Acad. Sci. USA 84: 7943-7947;Stockhaus et al., 1989, EMBO J. 8: 2445-2451) or, the CA/b-promoter(e.g., U.S. Pat. No. 5,656,496; U.S. Pat. No. 5,639,952; Barisal et al.,Proc. Natl. Acad. Sci. USA 89, (1992), 3654-3658) and the rubiscoSSU-Promoter (e.g., U.S. Pat. No. 5,034,322; U.S. Pat. No. 4,962,028) orfor a endosperm-specific expression the glutelin promoter (Leisy et al.,Plant Mol. Biol. 14,(1990), 41-50; Zheng et al., Plant J. 4,(1993),357-366; Yoshihara et al., FEBS Left. 383,(1996), 213-218), theshrunken-1 promotor (Werr et al., EMBO J. 4, (1985), 1373-1380), thewheat HMG 10 promotor, the USP promotor, the phaseolin promotor orpromoters from maize zein genes (Pedersen et al., Cell 29,(1982),1015-1026; Quatroccio et al., Plant Mol. Biol. 15 (1990), 81-93).

A termination sequence which terminates the nucleic acid moleculeaccording to the invention may serve to correctly end transcription andto add to the transcript a poly-A tail, which is considered to have afunction in stabilizing the transcripts. Such elements have beendescribed in the literature (cf. Gielen et al., 1989, EMBO J. 8:23-29)and are, as a rule, exchangeable as desired.

The nucleic acid molecules according to the invention or to be suitablyemployed in accordance with the invention can be used for generatingtransgenic plant cells and plants which show an increase and/orreduction in the activity of the soluble starch synthase III and of atleast one further enzyme of starch metabolism. To this end, the nucleicacid molecules according to the invention or to be suitably employed inaccordance with the invention are introduced into suitable vectors,provided with the regulatory nucleic acid sequences which are necessaryfor efficient transcription in plant cells, and introduced into plantcells. On the one hand, there is the possibility of using the nucleicacid molecules according to the invention or the nucleic acid moleculesto be suitably employed in accordance with the invention for inhibitingthe synthesis of the endogenous soluble starch synthase III and/or of atleast one further protein from the group A in the cells. This may beachieved with the aid of antisense constructs, in-vivo mutagenesis, acosuppression effect which occurs, or with the aid of suitablyconstructed ribozymes. On the other hand, the nucleic acid moleculesaccording to the invention or to be suitably employed in accordance withthe invention can be used for expressing the soluble starch synthase IIIand/or at least one further protein from the group A in cells oftransgenic plants and thus lead to an increased activity, in the cells;of the enzymes which have been expressed in each case.

In addition, there exists the possibility of using the nucleic acidmolecules according to the invention or to be suitably employed inaccordance with the invention for inhibiting the synthesis of theendogenous soluble starch synthase III and the overexpression of atleast one further protein from the group A in the cells. Finally, thenucleic acid molecules according to the invention or to be suitablyemployed in accordance with the invention may also be used forexpressing the soluble starch synthase III and for inhibiting at leastone further protein from the group A in cells of transgenic plants. Thetwo last-mentioned embodiments of the invention thus lead, in the cells,to a simultaneous inhibition and increase in activity of the enzymeswhich are inhibited or expressed, respectively.

The invention furthermore relates to a vector comprising a nucleic acidmolecule according to the invention.

The term “vector” encompasses plasmids, cosmids, viruses, bacteriophagesand other vectors conventionally used in genetic engineering whichcontain the nucleic acid molecules according to the invention and whichare suitable for transforming cells. Such vectors are preferablysuitable for transforming plant cells. Especially preferably, theypermit integration of the nucleic acid molecules according to theinvention, if appropriate together with flanking regulatory regions,into the genome of the plant cell. Examples are binary vectors such aspBinAR or pBinB33, which can be employed in agrobacteria-mediated genetransfer.

In a preferred embodiment, the vector according to the invention isdistinguished by the fact that the nucleotide sequence encoding aprotein having the function of a soluble starch synthase III orfragments thereof is present in sense or in antisense orientation.

In a further preferred embodiment, the vector according to the inventionis distinguished by the fact that the nucleotide sequence which encodesone or more proteins selected from the group A or fragments thereof ispresent in sense or in antisense orientation.

In a further preferred embodiment, the vector according to the inventionis distinguished by the fact that the nucleotide sequence which encodesone or more proteins selected from the group A or fragments thereof ispresent in sense or in antisense orientation.

In yet a further preferred embodiment, the vector according to theinvention is distinguished by the fact that the nucleotide sequencewhich encodes a plurality of proteins selected from the group A orfragments thereof is present partly in sense and partly in antisenseorientation.

Very especially preferably, the vector according to the inventioncomprises one or more regulatory elements which ensure transcription orsynthesis of an RNA in a prokaryotic or eukaryotic cell.

In addition, it is possible to introduce, by means of customarytechniques of molecular biology (see, for example, Sambrook et al.,1989, Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold SpringHarbor Laboratory Press, Cold Spring Harbour, N.Y.), various mutationsinto the DNA sequences according to the invention or the DNA moleculesto be suitably employed in accordance with the invention, which leads tothe synthesis of proteins with biological properties which may bealtered. On the one hand, it is possible to generate deletion mutants inwhich sequences are generated, by progressive deletions from the 5′ orfrom the 3′ end of the encoding DNA sequences which lead to thesynthesis of analogously truncated proteins. For example, such deletionsat the 5′ end of the DNA sequence allow the targeted production ofenzymes which, due to the removal of the relevant transit or signalsequences, are no longer localized in their original (homologous)compartment, but in the cytosol, or which, due to the addition of othersignal sequences, are localized in one or more other (heterologous)compartments (for example plastid, vacuole, mitochondrion, apoplast).

On the other hand, it is also feasible to introduce point mutations inpositions where an altered amino acid sequence affects, for example, theenzyme activity or the regulation of the enzyme. Thus, it is possible togenerate, for example mutants which have an altered K_(M) or K_(CAT)value or which are no longer subject to the regulatory mechanismsnormally present in the cell, e.g., via allosteric regulation orcovalent modification.

For the genetic engineering manipulation in prokaryotic cells, the DNAsequences according to the invention or to be suitably employed inaccordance with the invention or fragments of these sequences can beintroduced into plasmids which permit mutagenesis or an altered sequenceby the recombination of DNA sequences. Base exchanges may be performedor natural or synthetic sequences may be added, with the aid of standardmethods in molecular biology (cf. Sambrook et at, 1989, loc.cit.). Tolink the DNA portions to each other, adaptors or linkers may be attachedto the portions. Furthermore, manipulations which provide suitablerestriction cleavage sites or which remove excessive DNA or restrictioncleavage sites which are no longer needed may be employed. Whereinsertions, deletions or substitutions are suitable, in-vitromutagenesis, primer repair, restriction or ligation may be employed. Theanalytical methods which are generally carried out are sequenceanalysis, restriction analysis and, if appropriate, other methods ofbiochemistry and molecular biology.

The invention furthermore relates to a transgenic host cell exhibitingan altered starch metabolism, in particular to prokaryotic or eukaryoticcells, preferably bacterial or plant (monocotyledonous ordicotyledonous) cells (for example of E.coli, Agrobacterium,Solananceae, Poideae, rye, barley, oats, corn, wheat sorghum and millet,sago, rice, peas, marrowfat peas, cassava, potatoes, tomatoes, oilseedrape/canola, soybeans, hemp, flax, sunflowers, cowpeas, mung beans,beans, bananas or arrowroot) which contain one or more nucleic acidmolecules according to the invention or one or more vectors according tothe invention, or which is derived from such a cell.

Yet another subject of the invention is a transgenic host cellexhibiting an altered starch metabolism, in particular prokaryotic oreukaryotic cells, preferably bacterial or plant cells (for example ofE.coli, Agrobacterium, Solananceae, Poideae, rye, barley, oats, corn,wheat, sorghum and millet, sago, rice, peas, marrowfat peas, cassava,potatoes, tomatoes, oilseed rape/canola, soybeans, hemp, flax,sunflowers, cowpeas, mung beans, beans, bananas or arrowroot) comprisinga) at least one nucleotide sequence encoding a protein having thefunction of a soluble starch synthase III or fragments thereof, and b)one or more nucleotide sequences encoding a protein selected from thegroup A or fragments thereof or nucleotide sequences which hybridizewith the said nucleic acid molecules or which is derived from such acell.

Host cells according to the invention cannot only be generated by usingthe nucleic acid molecules according to the invention, but also by meansof successive transformation (e.g., by so-called supertransformation),by employing a plurality of portions of the nucleotide sequenceaccording to the invention or a plurality of vectors comprising portionsof the nucleotide sequence according to the invention which encode aprotein having the function of a soluble starch synthase III andadditionally encode one or more proteins selected from the group Aconsisting of branching enzymes, ADP glucose pyrophosphorylases,granule-bound starch synthases, soluble starch synthases I, II or other,debranching enzymes, disproportioning enzymes, plastidic starchphosphorylases, R1 enzymes, fragments thereof, and nucleic acidmolecules which hybridize with one of said nucleotide sequences orfragments thereof, in a plurality of cell transformation steps. Hereby,the cell is subsequently or simultaneously transformed in any order witha) at least one nucleic acid molecule encoding a protein having thefunction of a soluble starch synthase III, a fragment thereof or avector comprising said nucleic acid molecule and b) one or more nucleicacid molecules encoding a protein selected from the group A consistingof branching enzymes, ADP glucose pyrophosphorylases, granule-boundstarch synthases, soluble starch synthases I, II or other, debranchingenzymes, disproportioning enzymes, plastidic starch phosphorylases, R1enzymes; or fragments thereof,—preferably soluble starch syntheses I,soluble starch syntheses II and/or branching enzymes or fragmentsthereof—, and nucleic acid molecules which hybridize with one of saidnucleotide sequences or one or more vectors comprising one or more ofthe said nucleic acid molecules.

Another object of the invention is a method of generating a transgenichost cell, bacterial cell, plant cell or plant which synthesizes amodified starch, which comprises integrating into the genome of a cella) at least one nucleic acid molecule encoding a protein having thefunction of a soluble starch synthase III, a fragment thereof or avector comprising said nucleic acid molecule and b) one or more nucleicacid molecules encoding a protein selected from the group A consistingof branching enzymes, ADP glucose pyrophosphorylases, granule-boundstarch synthases, soluble starch synthases I, II or other, debranchingenzymes, disproportioning enzymes, plastidic starch phosphorylases, andR1 enzymes, or fragments thereof,—preferably, soluble starch synthasesI, soluble starch synthases II and/or branching enzymes or fragmentsthereof—, and nucleic acid molecules which hybridize with one of saidnucleotide sequences or one or more vectors comprising one or more ofthe said nucleic acid molecules.

A further embodiment of the present invention relates to a method ofgenerating a transgenic host cell, bacterial cell, plant cell or plantwhich synthesizes a modified starch, which comprises integrating one ormore nucleic acid molecules according to the invention or one or morevectors according to the invention into the genome of a cell.

Providing the nucleic acid molecules according to the invention makes itpossible to engage in the starch metabolism of plants, with the aid ofgenetic engineering methods, and to alter it in such a way that theresult is the synthesis of a modified starch which is altered relativeto starch synthesized in wild-type plants with regard to, for example,structure, water content, protein content, lipid content, fiber content,ash/phosphate content, amylose/amylopectin ratio, molecular massdistribution, degree of branching, granule size, granule shape andcrystallization, or else in its physico-chemical properties such asflowing and absorption behavior, gelatinization temperature, viscosity,thickening capacity, solubility, gel structure, transparency, thermalstability, shear stability, stability to acids, tendency to undergoretrogradation, gelling, freeze-thaw-stability, complex formation,iodine binding, film formation, adhesion power, enzyme stability,digestibility or reactivity.

There is also the possibility of increasing the yield in suitablygenetically engineered plants by increasing the activity of proteinswhich are involved in the starch metabolism, for example byoverexpressing suitable nucleic acid molecules, or by providing mutantswhich are no longer subject to the cell's regulatory mechanisms and/orwhich exhibit different temperature dependencies relating to theiractivity. A particularly pronounced increase in yield may be the resultof increasing the activity of one or more proteins which are involved inthe starch metabolism in specific cells of the starch-storing tissues oftransformed plants such as, for example, in the tuber in the case ofpotatoes or in the endosperm of corn or wheat. The economic importanceand the advantages of these possibilities of engaging in the starchmetabolism are enormous.

When expressing the nucleic acid molecules according to the invention orto be suitably employed in accordance with the invention in plants, itis possible, in principle, for the synthesized protein to be localizedin any desired compartment of the plant cell. To achieve location in aparticular compartment (cytosol, vacuole, apoplast, plastids,mitochondria), the transit or signal sequence which ensures locationmust, if necessary, be deleted (removed) and the remaining encodingregion must, if necessary, be linked to DNA sequences which ensurelocation in the particular compartment. Such sequences are known (see,for example, Braun et al., EMBO J. 11 (1992), 3219-3227; Wolter et al.,Proc. Natl. Acad. Sci. USA 85 (1988), 846-850; Sonnewald et al., PlantJ. 1 (1991), 95-106). To ensure the location in a particular plant partor tissue specific promoters may be used which are well known to theperson skilled in the art.

The generation of plant cells with a reduced activity of a proteininvolved in the starch metabolism can be achieved, for example, byexpressing a suitable antisense RNA, a sense RNA for achieving acosuppression effect, in-vivo mutagenesis or by expressing a suitablyconstructed ribozyme which specifically cleaves transcripts which encodeone of the proteins involved in the starch metabolism, using a nucleicacid molecule according to the invention, preferably by expressing anantisense transcript.

To this end, it is possible to use, on the one hand, a DNA moleculewhich encompasses all of the sequence which encodes a protein involvedin the starch metabolism inclusive of any flanking sequences which maybe present, as well as DNA molecules which only encompass fragments ofthe encoding sequence, these fragments having a minimum length of 15 bp,preferably of at least 100-500 bp, in particular over 500 bp. As a rule,DNA molecules are used which are shorter than 5000 bp, preferablyshorter than 2500 bp.

It is also possible to use DNA sequences which exhibit a high degree ofhomology to the sequences of the DNA molecules according to theinvention, but are not fully identical with them. The minimum homologyshould exceed approx. 65%. The use of sequences with a homology ofapproximately 75 to 85% and, in particular, approximately 85 to 95%, ispreferred.

The expression of ribozymes for reducing the activity of specificproteins in cells is known to those skilled in the art and described,for example, in EP-B 1-0 321 201. The expression of ribozymes in plantcells was described, for example, by Feyter et al. (Mol. Gen. Genet. 250(1996), 329-338).

Further, the proteins involved in the starch metabolism may also bereduced in the plant cells according to the invention by so-called“in-vivo mutagenesis”, where a hybrid RNA-DNA-oligonucleotide(“chimeroplast”) is introduced into cells by means of transforming them(Kipp P. B. et al., Poster Session at the 5^(th) International Congressof Plant Molecular Biology, 21-27, September 1997, Singapore; R. A.Dixon and C. J. Arntezen, Meeting report on “Metabolic Engineering inTransgenic Plants”, Keystone Symposia, Copper Mountain, Colo., USA,TIBITECH 15 (1997), 441-447; International Patent Application WO95/15972; Kren et al., Hepatology 25 (1997), 1462-1468; Cole-Strauss etal., Science 273 (1996), 1386-1389).

A fragment of the DNA component of the RNA-DNA-oligonucleotide used forthis purpose is homologous to a nucleic acid sequence of an endogenousprotein, but exhibits a mutation in comparison with the nucleic acidsequence of the endogenous protein or comprises a heterologous regionenclosed by the homologous regions.

Base pairing of the homologous regions of the RNA-DNA-oligonucleotideand of the endogenous nucleic acid molecule followed by homologousrecombination allows the mutation or heterologous region comprised inthe DNA component of the RNA-DNA-oligonucleotide to be transferred intothe genome of a plant cell. This leads to a reduced activity of theprotein involved in the starch metabolism.

Alternatively, the enzyme activities which are involved, in the plantcells, in the starch metabolism may be reduced by a cosuppressioneffect. This method is described, for example, by Jorgensen (TrendsBiotechnol. 8 (1990), 340-344), Niebel et al., (Curr. Top. Microbiol.Immunol. 197 (1995), 91-103), Flavell et al. (Curr. Top. Microbiol.Immunol. 197 (1995). 43-46), Palaqui and Vaucheret (Plant. Mol. Biol. 29(1995), 149-159), Vaucheret et al., (Mol. Gen. Genet. 248 (1995),311-317), de Borne et al. (Mol. Gen. Genet. 243 (1994), 613-621).

To inhibit the synthesis, in the transformed plants, of a plurality ofenzymes involved in starch biosynthesis, it is possible to use DNAmolecules for transformation which simultaneously comprise, in antisenseorientation and under the control of a suitable promoter, a plurality ofregions which encode the relevant enzymes. Each sequence may be underthe control of its promoter, or, alternatively, the sequences can betranscribed by a joint promoter as a fusion, so that the synthesis ofthe proteins in question is inhibited to approximately the same or to adifferent extent. As regards the length of the individual encodingregions which are used in such a construct, what has already been saidabove for the generation of antisense constructs also applies here. Inprinciple, there is no upper limit for the number of antisense fragmentstranscribed starting from a promoter in such a DNA molecule. However,the resulting transcript should not, as a rule, exceed a length of 25kb, preferably 15 kb.

Therefore, the nucleic acid molecules according to the invention or tobe suitably employed in accordance with the invention make it possibleto transform plant cells and simultaneously to inhibit the synthesis ofa plurality of enzymes.

Moreover, it is possible to introduce the nucleic acid moleculesaccording to the invention or to be suitably employed in accordance withthe invention into traditional mutants which are deficient or defective,with regard to one or more starch biosynthesis genes (Shannon andGarwood, 1984,in Whistler, BeMiller and Paschall, Starch: Chemistry andTechnology, Academic Press, London, 2^(nd) Edition: 25-86). Thesedefects can relate, for example, to the following proteins:granule-bound starch synthase (GBSS I) and soluble starch synthases (§I, II, III or other), branching enzymes (BE I, Iia and IIb), debranchingenzymes (R-enzymes, isoamylases, pullulanases), disproportioning enzymesand plastidic starch phosphorylases.

The present invention thus also relates to transgenic plant cellsobtainable by a process according to the invention which have beentransformed with a vector or nucleic acid molecule according to theinvention or the nucleic acid molecules to be suitably employed inaccordance with the invention, and to transgenic plant cells or plantsderived from such transformed cells. The cells according to theinvention contain one or more nucleic acid molecules according to theinvention or to be suitably employed in accordance with the invention,this preferably being linked to one or more regulatory DNA elements (forexample promoter, enhancer, terminator) which ensure the transcriptionin plant cells, in particular a promoter. The cells according to theinvention can be distinguished from naturally occurring plant cells,inter alia, by the fact that they contain a nucleic acid moleculeaccording to the invention which does not occur naturally in thesecells, or by the fact that such a molecule exists integrated at alocation in the cell's genome where it does not occur otherwise, i.e. ina different genomic environment. Furthermore, the transgenic plant cellsaccording to the invention can be distinguished from naturally occurringplant cells by the fact that they contain at least one copy of a nucleicacid molecule according to the invention stably integrated into theirgenome, if appropriate in addition to copies of such a molecule whichoccur naturally in the cells, resp., the nucleic acid molecules to besuitably employed in accordance with the invention. If the nucleic acidmolecule(s) introduced into the cells is (are) additional copies tomolecules which already occur naturally in the cells, then the plantcells according to the invention can be distinguished from naturallyoccurring plant cells in particular by the fact that this additionalcopy, or these additional copies, is, or are localized at sites in thegenome at which it does not occur naturally, or they do not occurnaturally. This can be checked, for example, with the aid of a Southernblot analysis.

Preferred plant cells according to the invention are those in which theenzyme activity of individual enzymes which are involved in the starchmetabolism is increased and/or reduced by in each case at least 10%,especially preferably at least 30% and very especially preferably by atleast 50%.

Moreover, the plant cells according to the invention can bedistinguished from naturally occurring plant cells preferably by atleast one of the following features: if the nucleic acid molecule whichhas been introduced is heterologous relative to the plant cell, thetransgenic plant cells exhibit transcripts of the nucleic acid moleculeswhich have been introduced. These can be detected by, for example,northern blot analysis. For example, the plant cells according to theinvention contain one or more proteins encoded by a nucleic acidmolecule according to the invention, resp., the nucleic acid moleculesto be suitably employed in accordance with the invention, which has beenintroduced. This can be detected by, for example, immunological methods,in particular by a western blot analysis.

If the nucleic acid molecule according to the invention, resp., thenucleic acid molecules to be suitably employed in accordance with theinvention which has been introduced is homologous relative to the plantcell, the cells according to the invention can be distinguished fromnaturally occurring cells, for example, on the basis of the additionalexpression of nucleic acid molecules according to the invention, resp.,the nucleic acid molecules to be suitably employed in accordance withthe invention. For example, the transgenic plant cells contain more orfewer transcripts of the nucleic acid molecules according to theinvention, resp., the nucleic acid molecules to be suitably employed inaccordance with the invention. This can be detected by, for example,northern blot analysis. “More” or “fewer” in this context meanspreferably at least 10% more or fewer, preferably at least 20% more orfewer and especially preferably at least 50% more or fewer transcriptsthan corresponding untransformed cells. Furthermore, the cellspreferably exhibit a corresponding increase or decrease in the contentof the proteins encoded by the introduced nucleic acid molecules (atleast 10%, 20% or 50%).

The transgenic plant cells can be regenerated to entire plants bytechniques known to those skilled in the art. The plants obtainable byregenerating the transgenic plant cells according to the invention, andprocesses for the generation of transgenic plants by regenerating entireplants from the plant cells according to the invention, are also subjectmatter of the present invention. Another subject of the invention areplants which comprise the transgenic plant cells according to theinvention. In principle, the transgenic plants can be plants of anyspecies, i.e. not only monocotyledonous, but also dicotyledonous plants.The plants are preferably crop plants and starch-storing plants such as,for example, cereal species (rye, barley, oats, corn, wheat, sorghum andmillet, etc.), sago, rice, peas, marrowfat peas, cassava, potatoes,tomatoes, oilseed rape/canola, soybeans, hemp, flax, sunflowers,cowpeas, mung beans or arrowroot.

The invention also relates to propagation material of the plantsaccording to the invention, for example fruits, seeds, tubers, rootstocks, seedlings, cuttings, calli, protoplasts, cell cultures and thelike.

Altering the enzymatic activities of the enzymes involved in the starchmetabolism results in the synthesis, in the plants generated by theprocess according to the invention, of a starch with an alteredstructure.

A large number of cloning vectors is available for preparing theintroduction of foreign genes into higher plants, vectors which comprisea replication signal for E. coli and a marker gene for the selection oftransformed bacterial cells. Examples of such vectors are pBR322, pUCseries, M13 mp series, pAGYG184 and the like. The desired sequences canbe introduced into the vector at a suitable restriction cleavage site.The plasmid obtained is used for transforming E. coli cells. TransformedE. coli cells are grown in a suitable medium, and then harvested andlysed. The plasmid is recovered. The analytical methods forcharacterizing the plasmid DNA obtained are generally restrictionanalyses, gel electrophoreses and other methods of biochemistry andmolecular biology (Sambrook et al. loc.cit.). After each manipulation,the plasmid DNA can be cleaved and DNA fragments obtained linked withother DNA sequences. Each plasmid DNA sequence can be cloned into thesame or other plasmids.

A large number of techniques is available for introducing DNA into aplant host cell. These techniques encompass the transformation of plantcells with T-DNA using Agrobacterium tumefaciens or Agrobacteriumrhizogenes as transformants, protoplast fusion by means of polyethyleneglycol (PEG), injection, DNA electroporation, the introduction of DNA bymeans of the biolistic method, and other possibilities (Gene Transfer toPlants. pp. 24-29. eds.: Potrykus, I. and Spangenberg, G., SpringerVerlag Berlin Heidelberg 1995).

The injection and electroporation of DNA into plant cells require noparticular aspects of the plasmids or the DNA used. Simple plasmids suchas, for example, pUC derivatives can be used. However, if entire plantsare to be regenerated from such transformed cells, the presence of aselectable marker gene is required.

Depending on the method of introducing desired genes into the plantcell, further DNA sequences may be required. If, for example, the Ti orRi plasmid is used for transforming the plant cell, at least the rightborder, but frequently the right and left border of the Ti and Riplasmid T-DNA must be linked to the genes to be introduced as a flankingregion.

If agrobacteria are used for the transformation, the DNA to beintroduced must be cloned into specific plasmids, either into anintermediate vector or into a binary vector. The intermediate vectorscan be integrated into the agrobacterial Ti or Ri plasmid by homologousrecombination owing to sequences which are homologous to sequences inthe T-DNA. The former also contains the vir region, which is requiredfor the T-DNA transfer. Intermediate vectors cannot replicate inagrobacteria. The intermediate vector can be transferred to Agrobactedumtumefaciens by means of a helper plasmid (conjugation). Binary vectorsare capable of replication in E.coli and in agrobacteria. They contain aselection marker gene and a linker or polylinker which are framed by theleft and right T-DNA border region. They can be transformed directlyinto the agrobacteria (Holsters et al. (1978) Mol. Gen. Genet.163:181-187). The agrobacterium which acts as the host cell shouldcontain a plasmid carrying a vir region. The vir region is required fortransferring the T-DNA into the plant cell. Additional T-DNA may bepresent. The agrobacterium thus transformed is used for transformingplant cells.

The use of T-DNA for transforming plant cells has been researchedintensively and been described in EP 120516; Hoekema, In: The BinaryPlant Vector System Offsetdrukkerij Kanters B.V., Alblasserdam (1985),Chapter V; Fraley et al., Crit. Rev. Plant. Sci. 4: 1-46 and An et al.(1985) EMBO J. 4: 277-287.

To transfer the DNA into the plant cell, plant explants can expedientlybe cocultured with Agrobacterium tumefaciens or Agrobacteriumrhizogenes. Entire plants can then be regenerated from the infectedplant material (for example leaf section, stalk sections, roots, butalso protoplasts, or plant cells grown in suspension culture) in asuitable medium which can contain, for example, antibiotics or biocidesfor selecting transformed cells. The resulting plants can then beexamined for the presence of the DNA which has been introduced. Otherpossibilities of introducing foreign DNA using the biolistic method orby protoplast transformation are known (cf., for example, Willmitzer, L,1993 Transgenic plants. In: Biotechnology, A Multi-Volume ComprehensiveTreatise (H. J. Rehm, G. Reed, A. Pulhler, P. Stadler, eds.), Vol. 2,627-659, VCH Weinheim-New York-Basel-Cambridge).

While the transformation of dicotyledonous plants via Ti-plasmid vectorsystems with the aid of Agrobacterium tumefaciens is well established,more recent work suggests that even monocotyledonous plants are indeedaccessible to transformation by means of agrobacterium-based vectors(Chan et al., Plant Mol. Biol. 22 (1993), 491-506; Hiei et al., Plant J.6 (1994), 271-282).

Alternative systems for the transformation of monocotyledonous plantsare the transformation by means of the biolistic method, protoplasttransformation, the electroporation of partially permeabilized cells,and the introduction of DNA by means of glass fibers.

Specifically different methods have been described in the literature forthe transformation of corn (cf., for example, WO95/06128, EP 0 513 849;EP 0 465 875). EP 292 435 describes a method with the aid of whichfertile plants can be obtained starting from a mucus-free, friable,granular corn callus. In this context, Shillito et al. (Bio/Technology 7(1989), 581) have observed that the capability of regenerating fertileplants requires starting from callus suspension cultures from which adividing protoplast culture with the capability of regenerating plantscan be made. Prioli and Söndahl (Bio/Technology 7 (1989), 589) alsodescribe the regeneration and obtaining of fertile corn plants from cornprotoplasts.

Once the introduced DNA is integrated into the genome of the plant cell,it is, as a rule, stable therein and is also retained in the progeny ofthe originally transformed cell. It normally contains a selection markerwhich mediates the transformed plant cells resistance to a biocide or anantibiotic such as kanamycin, G 418, bleomycin, hygromycin orphosphinothricin and the like. The individual marker chosen shouldtherefore allow the selection of transformed cells over cells which lackthe introduced DNA.

Within the plant, the transformed cells grow in the customary manner(see, also, McCormick et al. (1986) Plant Cell Reports 5:81-84). Theresulting plants can be grown normally and hybridized with plants whichhave the same transformed germplasm or other germplasm. The resultinghybrids have the corresponding phenotypic features.

Two or more generations should be grown to ensure that the phenotypicfeature is stably retained and inherited. Also, seeds or the like shouldbe harvested to ensure that the phenotype in question or other featureshave been retained.

Due to the expression of a nucleic acid molecule according to theinvention, the transgenic plant cells and plants according to theinvention synthesize a starch whose, for example, physico-chemicalproperties are altered in comparison with starch synthesized inwild-type plants.

Yet a further subject of the invention is the starch obtainable from aplant cell according to the invention, a plant according to theinvention, its propagation material or a process according to theinvention.

Yet another subject of the invention is a process for the production ofstarch in a manner known per se, in which host cells, plant cells,plants, plant parts or propagation material according to the inventionis processed, resp., integrated into the process.

In a preferred embodiment, the starch according to the invention isdistinguished in that its phosphate content is reduced by at least 30%,preferably at least 50%, especially preferably at least 70% and veryespecially preferably at least 90% in comparison with a starchobtainable from an untransformed cell or plant (i.e. the wild type) andin that its glucan content (cf. Fraction 3 in Example No. 13) afterisoamylase treatment in the exclusion volume of an HPLC column systemcomposed of 2 TSK-gel 2000SW columns connected in series and one TSK-gel3000SW column in 10 mM sodium acetate, pH 3.0 (at a flow rate of 0.35ml/min as shown in Example No. 13) is increased by at least 50%,preferably at least 150%, especially preferably at least 300% and veryespecially preferably at least 500%.

In a further embodiment, the starch according to the invention isdistinguished in that its phosphate content is increased by at least10%, preferably at least 30%, and especially preferably at least 50% incomparison with a starch obtainable from an untransformed cell or plant(i.e. the wild type) and in that its glucan content (cf. Fraction 3 inExample No. 13) after isoamylase treatment in the exclusion volume of anHPLC column system composed of 2 TSK-gel 2000SW columns connected inseries and one TSK-gel 3000SW column in 10 mM sodium acetate, pH 3.0 (ata flow rate of 0.35 ml/min as shown in Example No. 13) is increased byat least 50%, preferably at least 150%, especially preferably at least300% and very especially preferably at least 500%. Processes forextracting the starch from plants or starch-storing plant organs areknown to those skilled in the art. processes for extracting starch fromcorn kernels are described, for example, by Eckhoff et al. (Cereal Chem.73 (1996) 54-57). As a rule, the extraction of maize starch on anindustrial scale is performed by wet milling. Moreover, processes forextracting the starch from a variety of starch-storing plants aredescribed, for example in “Starch: Chemistry and Technology (Editors:Whistler, BeMiller and Paschall (1994), 2nd Edition, Academic Press Inc.London Ltd; ISBN 0-12-746270-8; see, for example, Chapter XII, pages412-468; corn and sorghum starches: production; by Watson; Chapter XIII,pages 469-479: tapioca, arrowroot and sago starches: production; byCorbishley and Miller; Chapter XIV, pages 479-490: potato starch:production and uses; by Mitch; Chapter XV, pages 491 to 506: wheatstarch: production, modification and uses; by Knight and Oson; andChapter XVI, pages 507 to 528: rice starch: production and uses; byRohmer and Klem). Devices normally used in processes for extractingstarch from plant material are separators, decanters, hydrocyclones,spray driers and fluidized-bed driers.

A further embodiment of the present invention also includes the use ofthe starch according to the invention for industrial application,preferably for the production of foodstuffs, packaging materials ordisposable products.

The starch according to the invention can be chemically and/orphysically modified by processes known to those skilled in the art andis suitable, in its unmodified or modified form, for a variety ofapplications in the food or non-food sector.

In principle, the possible uses of the starch according to the inventioncan be divided into two important sectors. One sector encompasses thehydrolysis products of the starch, mainly glucose and glucose units,which are obtained by enzymatic or chemical methods. They are used asstarting material for other chemical modifications and processes such asfermentation. What may be important here is the simplicity andinexpensive design of a hydrolysis process as is currently performedessentially enzymatically using amyloglucosidase. What would be feasibleis a financial saving by using less enzyme. This could be caused byaltering the structure of the starch, for example increasing the surfacearea of the granule, better degradability by a lower degree ofbranching, or a sterical structure which limits the accessibility forthe enzymes employed.

The other sector in which the starch according to the invention can beused as a so-called native starch, due to its polymeric structure, canbe divided into two further fields of application:

1. The Food Industry

Starch is a traditional additive to a large number of foodstuffs inwhich its function is essentially to bind aqueous additives or to causean increased viscosity or else increased gelling. Importantcharacteristics are the flowing characteristics, the sorptivecharacteristics, the swelling temperature, the gelatinizationtemperature, the viscosity, the thickening power, starch solubility,transparency, gel structure, thermal stability, shear stability,stability to acids, the tendency to undergo retrogradation, thefilm-forming capacity, the freeze-thaw-stability, digestibility and theability of forming complexes with, for example, inorganic or organicions.

2. The Non-food Industry

In this important sector, starch is employed as auxiliary for variouspreparation processes or as an additive in products. When using starchas an auxiliary, mention must be made, in particular, of the paper andboard industry. Starch acts mainly for retardation purposes (retainingsolids), binding filler particles and fine, as a stiffener and fordehydration. Moreover, the advantageous properties of the starchregarding stiffness, hardness, sound, touch, luster, smoothness, bondingstrength and the surfaces.

2.1. Paper and Board Industry

Within the papermaking process, four fields of application must bedistinguished, i.e. surface, coating, mass and spraying. With 80% of theconsumption, the surface starch accounts usually for the greatest starchquantity, 8% are used as coating starch, 7% as mass starch and 5% asspraying starch.

The demands on starch with regard to surface treatment are essentiallyhigh whiteness, an adapted viscosity, highly-stable viscosity, good filmformation and low dust formation. When used for coating, the solidscontent, a suitable viscosity, a high binding capacity and a highpigment affinity play an important role. Of importance when used asadditive to the mass is rapid, uniform, loss-free distribution, highmechanical strength and complete retention in paper cloth. If the starchis used in the spraying sector, again, an adapted solids content, highviscosity and a high binding capacity are of importance.

2.2. The Adhesives Industry

An important field of application for starches is in the adhesivesindustry, where the potential uses can be divided into four subsections:the use as a pure starch paste, the use in starch pastes which have beentreated with specialty chemicals, the use of starch as additive tosynthetic resins and polymer dispersions, and the use of starches asextenders for synthetic adhesives. 90% of the starch-based adhesives areemployed in the sectors production of corrugated board, production ofpaper sacks and bags, production of composite materials for paper andaluminum, production of boxboard and gumming adhesive for envelopes,stamps and the like.

2.3. Textile Industry and Textile Care Products Industry

An important field of application for starches as auxiliaries andadditives is the sector production of textiles and textile careproducts. The following four fields of application must be distinguishedwithin the textile industry: the use of starch as sizing agent, i.e. asauxiliary for smoothing and strengthening the burring behavior as aprotection from the tensile forces applied during weaving, and forincreasing abrasion resistance during weaving, starch as a textilefinishing agent, in particular after quality-reducing pretreatments suchas bleaching, dyeing and the like, starch as thickener in thepreparation of dye pastes for preventing bleeding, and starch asadditive to warping agents for sewing yarns.

2.4. Construction Materials Industry

The fourth field of application is the use of starches as additives inconstruction materials. An example is the production of gypsumplasterboards, where the starch which is admixed to the gypsum slurrygelatinizes with the water, diffuses to the surface of the plaster coreand there binds the board to the core. Other fields of application arethe admixture to rendering and mineral fibers. In the case ofready-mixed concrete, starch products are employed for delaying binding.

2.5. Soil Stabilization

A limited market for starch products is the production of soilstabilizers, which are employed for the temporary protection of the soilparticles from water when the soil is disturbed artificially. Accordingto present knowledge, product combinations of starch and polymeremulsions equal the previously employed products with regard to theirerosion- and crust-reducing effect, but are markedly less expensive.

2.6. Use in Crop Protection Products and Fertilizers

One field of application for using starch is in crop protection productsfor altering the specific properties of the products. Thus, starches areemployed for improving the wettability of crop protection products andfertilizers, for the metered release of the active ingredients, forconverting liquid active ingredients, volatile active ingredients and/oractive ingredients with an offensive odor into microcrystalline, stable,shapable substances, for mixing incompatible compounds and for extendingthe duration of action by reducing decomposition.

2.7. Pharmaceuticals, Medicine and the Cosmetics Industry

Another field of application is the sector of the pharmaceuticals,medicine and cosmetics industry. In the pharmaceuticals industry,starches are employed as binders for tablets or for diluting the binderin capsules. Moreover, starches are used as tablet disintegrants, sincethey absorb fluids after swallowing and swell within a short time tosuch an extent that the active ingredient is liberated. Medicinallubricating powders and wound powders are starch-based for reasons ofquality. In the cosmetics sector, starches are employed, for example, ascarriers of powder additives such as fragrances and salicylic acid. Arelatively large field of application for starch is toothpaste.

2.8. Addition of Starch to Coal and Briquettes

A field of application for starch is as an additive to coal andbriquettes. With an addition of starch, coal can be agglomerated, orbriquetted, in terms of high quantity, thus preventing earlydecomposition of the briquettes. In the case of barbeque coal, thestarch addition amounts to between 4 and 6%, in the case of calorizedcoal to between 10 0.1 and 0.5%. Moreover, starches are gainingimportance as binders since the emission of noxious substances can bemarkedly reduced when starches are added to coal and briquettes.

2.9. Ore Slick and Coal Silt Preparation

Furthermore, starch can be employed as flocculant in the ore slick andcoal silt preparation.

2.10. Foundry Auxiliary

A further field of application is as an additive to foundry auxiliaries.Various casting processes require cores made with sands treated withbinders. The binder which is predominantly employed nowadays isbentonite, which is treated with modified starches, in most casesswellable starches. The purpose of adding starch is to increaseflowability and to improve the binding power. In addition, the swellablestarches can meet other demands of production engineering, such as beingcold-water-dispersible, rehydratable and readily miscible with sand andhaving high water binding capacity.

2.11. Use in the Rubber Industry

In the rubber industry, starch is employed for improving the technicaland visual quality. The reasons are the improvement of the surfaceluster, the improvement of handle and of appearance, and to this endstarch is scattered to the tacky gummed surfaces of rubber materialsprior to cold curing, and also the improvement of the rubber'sprintability.

2.12. Production of Leather Substitutes

Modified starches may furthermore also be sold for the production ofleather substitutes.

2.13. Starch in Synthetic Polymers

In the polymer sector, the following fields of application can beenvisaged: the use of starch degradation products in the processingprocess (the starch is only a filler, there is no direct bond betweenthe synthetic polymer and the starch) or, alternatively, the use ofstarch degradation products in the production of polymers (starch andpolymer form a stable bond).

The use of starch as a pure filler is not competitive in comparison withother substances such as talc. However, this is different when thespecific properties of starch make an impact and thus markedly alter thespectrum of characteristics of the end products. An example of this isthe use of starch products in the processing of thermoplasts, such aspolyethylene. Here, the starch and the synthetic polymer are combined bycoexpression in a ratio of 1:1 to give a masterbatch, from which variousproducts are produced together with granulated polyethylene, usingconventional process techniques. By using starch in polyethylene films,an increased substance permeability in the case of hollow bodies, animproved permeability for water vapor, an improved antistatic behavior,an improved antiblock behavior and an improved printability with aqueousinks can be achieved. The current disadvantages relate to theinsufficient transparency, the reduced tensile strength and a reducedelasticity.

Another possibility is the use of starch in polyurethane foams. Byadapting the starch derivatives and by process-engineering optimization,it is possible to control the reaction between synthetic polymers andthe starches' hydroxyl groups in a directed manner. This results inpolyurethane films which have the following spectrum of properties,owing to the use of starch: a reduced heat extension coefficient, areduced shrinking behavior, an improved pressure-tension behavior, anincrease in permeability for water vapor without altering the uptake ofwater, a reduced flammability and a reduced ultimate tensile strength,no drop formation of combustible parts, freedom from halogens andreduced aging. Disadvantages which still exist are a reducedprintability and a reduced impact strength.

Product development is currently no longer restricted to films. Solidpolymer products such as pots, slabs and dishes which contain a starchcontent of over 50% may also be prepared. Moreover, starch/polymermixtures are considered advantageous since their biodegradability ismuch higher.

Starch graft polymers have become exceedingly important owing to theirextremely high water binding capacity. They are products with a starchbackbone and a side chain of a synthetic monomer, grafted on followingthe principle of the free-radical chain mechanism. The currentlyavailable starch graft polymers are distinguished by a better bindingand retention capacity of up to 1000 g of water per g of starch combinedwith high viscosity. The fields of application for these superabsorbershave extended greatly in recent years and are, in the hygiene sector,the products diapers and pads and, in the agricultural sector, forexample seed coatings.

What is decisive for the application of novel, genetically engineeredstarches are, on the one hand, structure, water content, proteincontent, lipid content, fiber content, ash/phosphate content,amylose/amylopectin ratio, molecular mass distribution, degree ofbranching, granule size and granule shape and crystallization, and, onthe other hand, also the characteristics which affect the followingfeatures: flowing and sorption behavior, gelatinization temperature,viscosity, thickening power, solubility, gel structure, transparency,heat stability, shear stability, stability to acids, tendency to undergoretrogradation, gel formation, freeze-thaw stability, complex formation,iodine binding, film formation, adhesive power, enzyme stability,digestibility and reactivity.

The production of modified starches by means of genetic engineeringmethods can, on the one hand, alter the properties, for example of thestarch derived from the plant in such a way that other modifications bymeans of chemical or physical alterations are no longer required. On theother hand, starches which have been altered by genetic engineeringmethods may be subjected to further chemical modification, which leadsto further improvements in quality for some of the above-describedfields of application. These chemical modifications are known inprinciple. They are, in particular, modifications by heat and pressuretreatment, treatment with organic or inorganic acids, enzymatictreatment, oxidations or esterifications, which lead, for example, tothe formation of phosphate starches, nitrate starches, sulfate starches,xanthate starches, acetate starches and citrate starches. Moreover,mono- or polyhydric alcohols in the presence of strong acids may beemployed for preparing starch ethers, resulting in starch alkyl ethers,o-allyl ethers, hydroxyalkyl ethers, O-carboxylmethyl ethers,N-containing starch ethers, P-containing starch ethers, S-containingstarch ethers, crosslinked starches or starch graft polymers.

A use of the starches according to the invention is in industrialapplication, preferably for foodstuffs or the preparation of packagingmaterials and disposable articles.

The examples which follow serve to illustrate the invention andconstitute in no way a restriction.

Abbreviations:

-   BE branching enzyme-   bp base pair-   GBSS granule bound starch synthase-   IPTG isopropyl-β-D-thiogalactopyranoside-   SS soluble starch synthase-   PMSF phenylmethylsulfonyl fluoride

Media and solutions used in the examples:

20 x SSC 175.3 g NaCI; 88.2 g sodium citrate to 1000 ml withdouble-distilled H₂0 pH 7.0 with 10 N NaOH Buffer A 50 mM Tris-HCI pH8.0; 2.5 mM DTT; 2 mM EDTA; 0.4 mM PMSF; 10% glycerol; 0.1% sodiumdithionite Buffer B 50 mM Tris-HCI pH 7.6; 2.5 mM DTT; 2 mM EDTA BufferC 0.5 M sodium citrate pH 7.6; 50 mM Tris-HCI pH 7.6; 2.5 mM DTT 2 mMEDTA 10 x TBS 0.2 M Tris-HCI pH 7.5; 5.0 M NaCI 10 x TBST 10x TBS; 0.1%(v/v) Tween 20 Elution buffer 25 mM Tris pH 8.3; 250 mM glycine Dialysisbuffer 50 mM Tris-HCI pH 7.0; 50 mM NaCI; 2 mM EDTA; 14.7 mMbeta-mercaptoethanol; 0.5 mM PMSF Protein buffer 50 mM sodium phosphatebuffer pH 7.2; 10 mM EDTA; 0.5 mM PMSF; 14.7 mM beta mercaptoethanol

DESCRIPTION OF THE FIGURES

FIG. 1 represents a schematic RVA temperature profile (viscosity andtemperature vs. Time [min]),this with the viscosimetric parametersT=gelatinization temperature, temperature at the beginning ofgelatinization; Max specifies the maximum viscosity; Min specifies theminimum viscosity; Fin specifies the viscosity at the end of themeasurement; Set is the difference (D) of Min and Fin (setback).

FIG. 2 shows the side-chain distribution of the amylopectin samplesdetermined on the right by means of HPAEC-PAC (voltage [mV] vs. Time[min]) and, on the left, determined by gel permeation chromatography(current [nC] vs. Time [min]) A=control (wild-type No. 1); B=(asSSII,No. 7); C=(asSSIII, No. 8); D=(asSSII asSSIII, No. 13); E=(asSSIIasSSIII, No. 14)

The numbers given in brackets in the description of the figures relateto the numbers of the starch samples described in Tables 1 and 2.

The following methods were used in the examples:

1. Cloning Method

vector pBluescript II SK (Stratagene) was used for cloning into E. coli

For the transformation of plants, the gene constructions were clone dinto the binary vector pBinAR Hyg (Höfgen & Willmitzer, 1990, Plant Sci.66:221-230) and pBinB33-Hyg. 2. Bacterial Strains and Plasmids

The E. coli strain DH5 a (Bethesda Research Laboratories, Gaithersburgh,USA) was used for the Bluescript vector p Bluescript II KS (Stratagene)and for the pBinAR Hyg and pBinB33Hyg constructs. The E. coli strain XL1-Blue was used for the in vivo excision.

pBinAR

The plasmid pBinAR is a derivative of the binary vector plasmid pBin19(Bevan, 1984), which was constructed as follows:

A 529 bp fragment encompassing the nucleotides 6909-7437 of thecauliflower mosaic virus 35S promoter was isolated from plasmid pDH51 asan EcoRl/Kpnl fragment (Pietrzak et al., 1986), ligated between theEcoRI and Kpnl cleavage sites of the pUC18 polylinker and was termedplasmid pUC18-35S. With the aid of the restriction endonucleases HindIIIand PvuII, a 192 bp fragment was isolated from plasmid pAGV40(Herrera-Estrella et al., 1983), which encompasses DNA of the Ti plasmidpTiACH5 (Gielen et ai, 1984) (nucleotides 11749-11939). After the PvuIIcleavage site had been provided with SphI linkers, the fragment wasligated between the SphI and HindIII cleavage sites of pUC18-35S, andthis was termed plasmid pA7. Moreover, the entire polylinker comprisingthe 35S promoter and the ocs terminator was excised with EcoRI andHindIII and ligated into the suitably cleaved pBin19. This gave rise tothe plant expression vector pBinAR (Höfgen and Willmitzer, 1990).

pBinB33

The promoter of the Solanum tuberosum patatin gene 833 (Rocha-Sosa etal., 1989) was ligated, as a Dral fragment (nucleotides−1512-+14) intothe Sst I-cleaved vector pUC19, which had been made blunt-ended with theaid of T4-DNA polymerase. This gave rise to the plasmid pUC19-B33. TheB33 promoter was excised from this plasmid with EcoRI and SmaI andligated into the suitably cleaved vector pBinAR. This gave rise to theplant expression vector pBinB33.

pBinAR-Hyg

Starting from plasmid pA7 (cf. description of vector pBinAR), theEcoRI-HindIII fragment comprising the 35S promoter, the ocs terminatorand the polylinker portion between 35S promoter and ocs terminator wasintroduced into the suitably cleaved plasmid pBin-Hyg.

pBinB33-Hyg

Starting from plasmid pBinB33, the EcoRI-HindIII fragment comprising theB33 promoter, part of the polylinker and the ocs terminator was cleavedout and ligated into the suitably cleaved vector pBin-Hyg. This gaverise to the plant expression vector pBinB33-Hyg.

3. Transformation of Agrobacterium tumefaciens

The DNA was transferred by direct transformation following the method ofHofgen & Willmitzer (1988, Nucleic Acids Res. 16:9877). The plasmid DNAof transformed agrobacteria was isolated following the method of Bimboim& Doly (1979, Nucleic Acids Res. 7:1513-1523), subjected to suitablerestriction cleavage, and then analyzed by gel electrophoresis.

4. Transformation of Potatoes

The plasmids were transformed into the potato plants (Solanum tuberosumL.cv. Desiree, Vereinigte Saatzuchten eG, Ebstorf) with the aid of theAgrobacterium tumefaciens strain C58C1 (Dietze at al. (1995) in GeneTransfer to Plants. pp. 24-29, eds.: Potrykus, I. and Spangenberg, G.,Springer Verlag, Deblaere et al., 1985, Nucl. Acids Res. 13:4777-4788).

Ten Small leaves of a sterile potato culture which had been wounded witha scalpel were placed into 10 ml MS medium (Murashige & Skoog (1962)Physiol. Plant. 15: 473) supplemented with 2% sucrose and containing 50ml of an Agrobacterium tumefaciens overnight culture grown underselection conditions. After the culture had been shaken gently for 3-5minutes, it was incubated for 2 more days in the dark. For callusinduction, the leaves were then placed on MS medium supplemented with1.6% glucose, S mg/l naphthyl acetic acid, 0.2 mg/l benzylaminopurine,250 mg/l claforan, 50 mg/l canamycin, and 0.80% Bacto agar. After theleaves had been incubated for one week at 25° C. and 3000 Lux, they wereplaced on MS medium supplemented with 1.6% glucose, 1.4 mg/l zeatinribose, 20 mg/l naphthylacetic acid, 20 mg/l gibberellic acid, 250 mg/lclaforan, 50 mg/l canamycin, and 0.80% Bacto agar, to induce shoots.

5. Plant Culture Regime

Potato plants were kept in the greenhouse under the following regime:

Light period 16 h at 25,000 Lux and 22° C. Dark period 8 hours at 15° C.Atmospheric humidity 60%6. Radiolabeling of DNA Fragments

The DNA fragments were radiolabeled with the aid of a DNA Random PrimerLabeling Kit by Boehringer Mannheim (Germany) following themanufacturer's instructions.

7. Determination of Starch Synthase Activity

Determination of starch synthase activity was done by determining theincorporation of ¹⁴ C Glucose from ADP [¹⁴ C D glucose] into amethanol/KCI-insoluble product as described by Denyer & Smith, 1992,Planta 186:609-617.

8. Detection of Soluble Starch Synthases in the Native Gel

To detect the activity of soluble starch synthases by non-denaturing gelelectrophoresis, tissue samples of potato tubers were hydrolyzed in 50mM Tris-HCl pH 7.6, 2 mM DTT, 2.5 mM EDTA, 10% glycerol and 0.4 mM PMSF.The electrophoresis was carried out in a MiniProtean II chamber(BioRAD). The monomer concentration of the 1.5-mm-thick gels was 7.5%(w/v), and 25 mM Tris-glycine pH 8.4 was used as gel buffer and runningbuffer. Identical amounts of protein extract were applied and separatedfor 2 hours at 10 mA per gel.

The activity gels were subsequently incubated in 50 mM Tricine-NaOH pH8.5, 25 mM potassium acetate, 2 mM EDTA, 2 mM DTT, 1 mM ADP-glucose,0.1% (w/v) amylopectin and 0.5 M sodium citrate. The glucans formed werestained with Lugol's solution.

9. Starch Analysis

The starch formed by the transgenic potato plants was characterized bythe following methods:

a) Determination of the Amylose/Amylopectin Ratio in Starch From PotatoPlants

Starch was isolated from potato plants by standard methods, and theamylose:amylopectin ratio was determined by the method described byHovenkamp-Hermelink et al.(Potato Research 31 (1988) 241-246).

b) Determination of the Phosphate Content

In potato starch, some glucose units may be phosphorylated on the carbonatoms at positions C2, C3 and C6. To determine the degree ofphosphorylation at the C6-position of the glucose, 100 mg of starch werehydrolyzed for 4 hours at 95° C. in 1 ml of 0.7 M HCI (Nielsen et. al.(1994) Plant Physiol. 105: 111-117). After neutralization with 0.7 MKOH, 50 ml of the hydrolysate were subjected to a visual-enzymatic testto determine glucose-6-phosphate. The change in absorption of the testbatch (100 mM imidazole/HCI; 10 mM MgCI₂; 0.4 mM NAD; 2 units ofLuconostoc mesenteroides, glucose-6-phosphate dehydrogenase; 30° C.) wasmonitored at 334 nm. The total phosphate was determined as described byAmes, 1996. Methods in Enzymology VIII, 115-118.

c) Analysis of the Amylopectin Side Chains

To analyze distribution and length of the side chains in the starchsamples, 1 ml of a 0.1% starch solution was digested with 0.4 U ofisoamylase (Megazyme International Ireland Ltd., Bray, Ireland)overnight at 37° C. in 100 mM sodium citrate buffer, pH 3.5.

The rest of the analysis was carried out as described by Tomlinson etal., (1997), Plant J. 11 :31-47, unless otherwise specified.

d) Granule Size Determination

The granule size was determined using a “Lumosed” photosedimentometer byRetsch GmbH, Germany. To this end, 0.2 g of starch were suspended inapprox. 150 ml of water and immediately measured. The program suppliedby the manufacturer calculated the mean diameter of the starch granules,assuming an average starch density of 1.5 g/l.

e) Gelatinization Properties

The gelatinization or viscosity properties of the starch were recordedusing a Viskograph E by Brabender OHG, Germany, or using a Rapid ViscoAnalyzer, Newport Scientific Pty Ltd, Investment Support Group,Warriewood NSW 2102, Australia. When using the Viskograph E, asuspension of 20 g of starch in 450 ml of water was subjected to thefollowing heating program: heating at 3°/min from 50° C. to 95° C., keepconstant for 30 minutes, cool at 3°/min to 30° C., and again keepconstant for 30 minutes. The temperature profile gave characteristicgelatinization properties.

When measuring using the Rapid Visco Analyzer (RVA), a suspension of 2 gof starch in 25 ml of water was subjected to the following heatingprogram: suspend for 60 seconds at 50° C., heat at 12°/min from 50° C.to 95° C., keep constant for 2.5 minutes, cool at 12°/min to 50° C., andagain keep constant for 2 minutes. The RVA temperature profile gave thevisco metric parameters of the test starches for the maximum viscosity(Max), the end viscosity (Fin), the gelatinization temperature (T), theminimum viscosity occurring after the maximum viscosity (Min), and thedifference between minimum and end viscosity (setback, Set) (cf. Table 1and FIG. 1).

f) Determination of the Gel Strength

To determine the gel strength by means of a Texture Analyzer, 2 g ofstarch were gelatinized in 25 ml of water (cf. RVA measurement) and thenstored for 24 hours in a sealed container at 25° C. with the exclusionof air. The samples were mounted underneath the probe (circular stamp)of a Texture Analyzer TA-XT2 (Stable Micro Systems), and the gelstrength was determined with the following parameter settings:

Test speed 0.5 mm Penetration depth 7 mm Contact area (of the stamp) 113mm² Pressure/contact area 2 g10. Determination of Glucose, Fructose and Sucrose

To determine the glucose, fructose and sucrose content, Small tuberportions (approx. diameter 10 mm) of potato tubers were frozen in liquidnitrogen and then extracted for 30 minutes at 80° C. in 0.5 ml of 10 mMHEPES, pH 7.5; 80% (vol./vol.) ethanol. The supernatant, which containsthe solubles: was removed and the volume was determined. The supernatantwas used for determining the amount of soluble sugars. The quantitativedetermination of soluble glucose, fructose and sucrose was carried outin a batch with the following composition

100,0    mM imidazole/HCl, pH 6.9 1.5 mM MgCl₂ 0.5 mM NADP⁺ 1.3 mM ATP10-50 μl sample 1.0 unit yeast glucose-6-phosphate dehydrogenase

The batch was incubated for 5 minutes at room temperature. The sugarswere subsequently determined photometrically by measuring the absorptionat 340 nm after the successive addition of

1.0 unit yeast hexokinase (to determine glucose),

1.0 unit yeast phosphoglucoisomerase (to determine fructose) and

1.0 unit yeast invertase (to determine sucrose)

11. Determination of the Water Uptake Capacity (WUC)

To determine the water uptake capacity, the solubles of the starch whichhad swelled at 70° C. were removed by centrifugation (10 min at10,000×g) and the residue was then weighed. The water uptake capacity ofthe starch was based on the initial starch quantity corrected by thesoluble matter.WUC (g/g)=(residue−(initial quantity−solubles))/(initialquantity−solubles)

EXAMPLES Example 1 Preparation of Plasmid p35SαSSI-Hyg

A 1831 bp Asp718/XbaI fragment containing a partial cDNA encoding thepotato SS I (Abel, G., (1995), PhD thesis, Freie Universitat Berlin) wasintroduced between the Asp718 and XbaI cleavage site of the vectorpBinAR-Hyg in antisense orientation relative to the 35S promoter.

Example 2 Preparation of Plasmid p35S-SSI-Kan

A 2384 bp EcoRI fragment containing cDNA encoding potato S8 I (Abel1995, loc.cit.) was made blunt-ended and introduced into the vectorpBinAR, which had previously been cut with SmaI, in sense orientationrelative to the 35S promoter.

Example 3 Preparation of Plasmid p35αSSII-Kan

A 1959 bp SmaI/Asp718 fragment containing a partial cDNA encoding potatoSS II (Abel, 1995, termed GBSS II therein) was made blunt-ended andintroduced into the SmaI cleavage site of the vector pBinAR in antisenseorientation relative to the 35S promoter.

Example 4 Preparation of Plasmid pB33-SSII-Hyg

A 2619 bp SmaI/SalI fragment containing a cDNA encoding the potato SS II(Abel, 1995, loc.cit.) was introduced into the vector pBinB33-Hyg, whichhad previously been cut with SmaI and SalI in sense orientation relativeto the B33 promoter.

Example 5 Preparation of Plasmid p35SαSSIII-Hyg

A 4212 bp Asp718/Xbal fragment containing a cDNA encoding the potato SSIII (Abel et al., 1996, Plant J. 10(6):981-991) was inserted between theAsp718 and the XbaI cleavage site of the vector pBinAR-Hyg in antisenseorientation relative to the 35S promoter.

Example 6 Preparation of Plasmid p35S-SSIII-Kan

A 4191 bp EcoRI fragment containing a cDNA encoding the potato SS III(Abel et al., 1996, loc.cit.) was made blunt-ended and introduced intothe SmaI cleavage site of the vector pBinAR in sense orientationrelative to the 35S promoter.

Example 7 Preparation of Plasmid pB33αBEαSSIII-Kan

A 1650 bp Hindll fragment which contains a partial cDNA encoding thepotato BE enzyme (Kossmann et al., 1991, Mol. & Gen. Genetics230(1-2):39-44) was made blunt-ended and introduced into the vectorpBinB33 which had bneen precut with SmaI in antisense orientationrelative to the B33 promoter. The resulting plasmid was cut open withBamHI. A 1362 bp BamHI fragment containing a partial cDNA encoding thepotato SS III enzyme (Abel et al., 1996, loc.cit.) was introduced intothe cleavage site, again in antisense orientation relative to the B33promoter.

Example 8 Preparation of Plasmid p35SαSSII-αSSIII-Kan

A 1546 bp EcoRV/HincII fragment containing a partial cDNA encoding forthe potato SS II (Abel, 1995, loc.cit.) was cloned into the vectorpBluescript II KS which had been cut with EcoRV/HincII, then excised viaan Asp718/BamHI digest and introduced in antisense orientation relativeto the 35S promoter into the vector pBinAR which had been digested inthe same manner. Then, a 1356 bp BamHI fragment containing a partialcDNA encoding the potato SS III (Abel et al., 1996, loc.cit.) wasintroduced into the BamHI cleavage site of the vector pBinAR-SSII, againin antisense orientation relative to the 35S promoter.

Example 9 Preparation of Plasmid pB33 αSSlαSSIII-Kan

A 2384 bp EcoRI fragment containing a cDNA encoding the potato SS I(Abel, 1995, loc.cit.) was made blunt-ended and cloned into theSmaI-cleavage site of the pBinB33 vector in antisense orientationrelative to the B33 promoter. A 1362 bp BamHI fragment containing apartial cDNA encoding the potato SS III (Abel et al., 1996, loc.cit.)was introduced into the BamHI cleavage site of the resulting vector,again in antisense orientation relative to the B33 promoter.

Example 10 Preparation of Plasmid p35SαSSII-Hyg

A 1959 bp SmaI/Asp718 fragment containing a partial cDNA encoding forthe SS II (Abel, 1995, loc.cit.) was made blunt-ended and introducedinto the SmaI cleavage site of the pBinAR-Hyg vector in antisenseorientation relative to the 35S promoter.

Example 10B Preparation of Plasmid pB33 αR1-Hyg

A 1.9 kB Asp718 fragment containing a partial cDNA encoding R1 proteinderived from s. tuberosum (WO 97/11188) was obtained from the vectorpBluescript. The fragment was cloned into the Asp718 restriction siteafter the B33 promoter in anti-sense orientation relative to the vectorpB33-Binar-Hyg comprising hygromycin resistance.

Example 11 Introduction of the Plasmids Into the Genome of Potato Cells

The plasmids given in Examples 1 to 10 were transferred intoagrobacteria, either individually and/or in succession, with the aid ofwhich potato cells were transformed as described above. Subsequently,entire plants were regenerated from the transformed plant cells.

Transgenic plant cells of the genotype asSSI-asSSII-asSSIII weregenerated by transformation with the plasmid p35SαSSI-Hyg described inEx. No. 1 and subsequent retransformation with the plasmidp35SαSSII-αSSIII-Kan described in Ex. No. 8.

Transgenic plant cells of the genotype asSSII-asSSI-asSSIII weregenerated by transformation with the plasmid p35SαSSII-Hyg described inEx. No. 10 and subsequent retransformation with the plasmidpB33αSSlαSSIII-Kan described in Ex. No. 9.

As a result of the transformation, the transgenic potato plantssynthesized altered starch varieties.

Example 12 Physico-chemical Characterization of the Modified Starches

The starch formed by the transgenic plants generated in accordance withExample 11 differs, for example, from starch synthesized by wild-typeplants (potato) with regard to its phosphate or amylose content and theviscosities and gelatinization properties which were determined by RVA.The results of the physico-chemical characterization of the modifiedstarches are shown in Table 1. In the antisense constructs, the enzymeactivities of the suppressed soluble starch synthases were reduced by upto 85% relative to the untransformed control plants.

TABLE 1 Properties of the modified starches RVA RVA RVA RVA GelPhosphate Amylose Max Min Fin Set RVA T strength No. Genotype (%) (%)(%) (%) (%) (%) (%) (%)  1 Désirée (wild 100 100 100 100 100 100 100 100type)  2 asSSI 100 100 113 100 100 114 100 112  3 oeSSI 140 100 118 152111 45 100 106  4 oeSSI 91 100 87 178 131 55 100 335  5 oeSSI 127 100100 157 121 63 100 313  6 cosSSI 100 100 106 100 100 100 100 127  7asSSIII 55 118 76 91 95 113 98 151  8 asSSIII 197 123 82 75 76 79 95 84 9 oeSSIII 100 100 100 88 87 100 68 10 cosSSIII 210 100 60 70 74 95 8311 asBE 170 91 124 94 90 76 100 91 12 asBE-asSSIII 292 128 69 75 97 95100 13 asSSII-asSSIII 31 124 30 77 107 229 93 212 14 asSSII-asSSIII 39110 45 88 113 216 94 189 15 asSSI-asSSIII 115 15b asSSI-asSSIII 86 10082 74 96 168 100 100 16 asSSII-asSSIII- asSSI 17 asSSI-asSSii- 54 115 60141 105 133 97 105 asSSIII 18 asSSII-asSSI- asSSIII 19 asBE-asSSIII- 37085 131 55 60 84 93 66 oeSSI 20 asBE-asSSIII- 125 136 asR1 21oeSSIII-oeSSII 105 100 127 122 126 136 94 189 Key: SSI = starch synthaseisoform I; SSII = starch synthase isoform II; SSIII = starch synthaseisoform III; BE = branching enzyme; as = antisense; oe = overexpressed(sense); cos = cosuppressed (sense); Rapid Visco Analyzer—(RVA) data:Max = maximum viscosity; Min = minimum viscosity; Fin = viscosity at theend of the measurement; Set is the difference (D) of Min and Fin(setback); T = gelatinization temperature The percentages are based onthe wild type (=100%).

Example 13 Characterization of the Side Chains of the Modified Starches

The glucan chains were separated after removing the amylose by means ofthymol precipitation (Tomlinson et al. loc. cit.) using a highperformance anion exchanger chromatography system with an amperometricdetector (HPEAC-PAD, Dionex). The samples (10 mg/ml amylopectin) weredissolved in 40% DMSO and 1/10 part by volume of 100 mM sodium acetatepH 3.5 and 0.4 U of isoamylase (Megazyme) were added. After incubation,10 μl of the sample were applied to the column system and eluted asdescribed by Tomlinson et al. (loc. cit.).

The results of the HPEAC-PAD analysis regarding length and distributionof the side chains of the starch samples Nos. 1, 7, 8, 13 and 14 (cf.Tables 1 and 2) are shown in FIG. 2.

Another HPLC system for detecting the side-chain distribution consistedof 3 columns connected in series (2 TSK-Gel 2000SW and one TSK-Gel3000SW, TosoHaas, Stuttgart, Germany) as described by Hizukuri ((1986)Carbohydr. Res. 147:342-347). 100 μI of the prepared sample were appliedto the column system. The eluent used was 10 mM sodium acetate pH 3.0 ata flow rate of 0.35 ml/min. The glucans were detected by means of arefraction index detector (Gynkotek), and the chain lengths of theeluted linear glucans were determined by mass spectrometry and iodometry(Hizukuri (1986) loc.cit.).

The results of the gel-chromatographic HPLC analysis regarding lengthand distribution of the side chains of starch samples Nos. 1, 7, 8, 13and 14 (cf. Tables 1 and 2) are shown in FIG. 2.

Table 2 shows the percentages of various side-chain fractions of thestarches which have been analyzed. Fraction 1 represents the percentageof the A and B1 chains (Hizukuri (1986) loc.cit.), Fraction 2 representsthe percentage of the B2, B3 and B4 chains (Hizukuri (1986) loc.cit.)and Fraction 3 shows the percentage of the high-molecular glucanmolecules which elute in the exclusion volume.

TABLE 2 Distribution of the amylopectin side chains of the modifiedstarch No. Genotype Fraction 1 (%) Fraction 2 (%) Fraction 3 (%) 1Désirée 58.7 40.3 1.0 (wild type) 7 asSSII 62.6 36.5 0.9 8 asSSIII 72.426.3 1.3 13 asSSIIasSSIII 66.9 27.5 5.6 14 asSSIIasSSIII 61.5 35.1 3.4

1. An isolated recombinant nucleic acid molecule comprising a. at leastone nucleotide sequence encoding a potato soluble starch synthase III;and b. one or more nucleotide sequences which encode a potato solublestarch synthase II, wherein said isolated recombinant nucleic acidmolecule inhibits the synthesis of endogenous soluble starch synthaseIII and endogenous soluble starch synthase II when introduced intoplants.
 2. The nucleic acid molecule of claim 1, wherein said nucleicacid molecule is a deoxyribonucleic acid molecule.
 3. The nucleic acidmolecule of claim 2, wherein said nucleic acid molecule is a cDNAmolecule.
 4. The nucleic acid molecule of claim 1, wherein said nucleicacid molecule is a ribonucleic acid molecule.
 5. An isolated vectorcomprising the nucleic acid molecule of claim
 1. 6. The vector of claim5, wherein said nucleotide sequence encoding potato soluble starchsynthase III is present in sense orientation.
 7. The vector of claim 5,wherein said nucleotide sequence encoding potato soluble starch synthaseIII is present in antisense orientation.
 8. The vector of claim 5,wherein said nucleotide sequence encoding potato soluble starch synthaseII is present in sense orientation.
 9. The vector of claim 5, whereinsaid nucleotide sequence encoding potato soluble starch synthase II ispresent in antisense orientation.
 10. The vector of claim 5, whereinsaid nucleic acid molecule being linked to one or more regulatoryelements which ensure transcription and synthesis of an RNA in aeukaryotic cell.
 11. A transgenic plant cell, comprising a) at least onenucleotide sequence encoding a potato soluble starch synthase III; andb) one or more nucleotide sequences encoding a potato soluble starchsynthase II, wherein said nucleotide sequences inhibit the synthesis ofendogenous soluble starch synthase III and endogenous soluble starchsynthase II.
 12. A method of generating a transgenic plant whichsynthesizes a modified starch, comprising the step of regenerating anentire plant from a plant cell of claim
 11. 13. A transgenic plantobtainable according to the method of claim 12, wherein said plantcomprises said plant cell.
 14. The plant of claim 13, wherein said plantis a starch-storing plant.
 15. The plant of claim 13, wherein said plantis potato.
 16. Propagation material of the plant of claim 13, whereinsaid propagation material contains said plant cell.
 17. A method for thepreparation of transgenic plant cells comprising the step oftransfecting plant cells with the nucleic acid molecule of claim
 1. 18.A method for producing starch comprising the step of obtaining starchfrom a plant cell as claimed in claim
 11. 19. A method for thepreparation of a transgenic plant cell comprising transfecting a plantcell with the vector of claim
 5. 20. A method of generating a transgenicplant cell comprising simultaneously or subsequently integrating intothe genome of a plant cell a) at least one nucleotide sequence encodinga potato soluble starch synthase III; and b) one or more nucleotidesequences encoding a potato soluble starch synthase II, therebyinhibiting the synthesis of endogenous soluble starch synthase III andendogenous soluble starch synthase II enzyme.
 21. A method for producingstarch comprising the step of obtaining starch from a plant as claimedin claim 15.